WO2022200572A1

WO2022200572A1 - Use of d-enantiomeric peptide ligands of monomeric tau for the therapy of various tauopathies

Info

Publication number: WO2022200572A1
Application number: PCT/EP2022/057923
Authority: WO
Inventors: Dieter Willbold; Jeannine Mohrlüder; Marc Sevenich; Karoline Bianka Santur; Tim Altendorf
Original assignee: Forschungszentrum Jülich GmbH
Priority date: 2021-03-25
Filing date: 2022-03-25
Publication date: 2022-09-29
Also published as: DE102021107546A1; EP4313301A1

Abstract

The invention relates to a peptide, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, or SEQ ID NO. 2, or SEQ ID NO. 3, or SEQ ID NO. 4, or SEQ ID NO. 5, or SEQ ID NO. 6, or SEQ ID NO. 7 and homologues, fragments and parts thereof, and to such a peptide for use in the treatment of tauopathies.

Description

USE OF D-ENANTIOMERIC PEPTIDE LIGANDS OF MONOMERIC TAU FOR THERAPY OF VARIOUS TAUOPATHIES

description

The invention relates to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6 or SEQ ID NO: 7, homologues, fragments and parts thereof and such a peptide for use in the treatment of tauopathies.

Alzheimer's dementia (AD) is an estimated proportion of almost two thirds - which corresponds to about 27 million people - the most common neurodegenerative disease worldwide. It is assigned to a heterogeneous group of different tauopathies, which are symptomatically characterized by progressive dementia. The intraneuronal deposits that occur in the brain, which contain amyloid structures of the hyperphosphorylated tau protein, usually correlate with the extent of cognitive deficits in the course of the disease.

As a neuronal microtubule-associated protein, tau is significantly involved in the assembly and stabilization of microtubules. Hyperphosphorylation of tau triggers its misfolding and aggregation up to the formation of so-called neurofibrillary tangles, which are a neuropathological hallmark of most tauopathies. In the brain adult humans express six tau isoforms by alternative splicing, which are between 352 and 441 amino acids long. Depending on the composition of isoforms present, different incorrect conformations can be formed, which are specific for the present tau pathology.

Toxic, self-replicating, and propagating tau aggregates are hallmarks of various tauopathies. In these pathologies, there is a characteristic prion-like proliferation of misfolded tau aggregates in the CNS, which is associated with neurodegeneration of the affected brain areas. Several studies have demonstrated the uptake of tau aggregates by cells, "seeding" effects (aggregates that induce the conversion of monomeric to misfolded, oligomeric or fibrillar tau as a template), as well as tau aggregate transfer between cell cultures Pathology reflect neuroanatomically connected brain regions.

To date, there is no active substance or medication that counteracts the causes of AD. The drugs used and approved to date alleviate some of the symptoms associated with Alzheimer's dementia. However, they are unable to slow down the progression of the disease or bring about a cure. There are some substances that have shown success in preventing AD in animal experiments, but not (necessarily) in treating AD.

However, five drugs (Aricept, Exelon, Razadyne, Donepezil, and Namenda) are available that relieve the symptoms of the disease and make everyday life easier for those affected.

Frontotemporal dementia cannot be treated causally either, so that the course of the disease can neither be slowed down nor stopped. First and foremost, antidepressants are used, which can have a symptom-relieving effect.

So far, there is no way to stop or slow down the pathology of progressive supranuclear palsy. Some symptoms do not respond to any medication. Antidepressants (such as Prozac, Elavil, and Tofranil) are used to relieve other symptoms, and special glasses and walking aids are used to make everyday life easier. The subacute sclerosing panencephalitis caused by a measles infection cannot be cured at the present time. Antiviral therapeutic agents (isoprinosine and ribavirin) as well as immunomodulating substances (interferon alpha) can stop the progression of the disease and increase the life expectancy of the patients. However, the side effects of long-term treatment are not yet known.

So far, there is no drug available for corticobasal degeneration that slows down or stops the progression of the disease. The symptoms of corticobasal degeneration are usually therapy-resistant. The substance clonazepam is used against myoclonus. Physiotherapy and speech therapy can help to cope with everyday life.

In conclusion, a causative and significantly life-prolonging therapy is not available for most, if not all, tauopathies and is urgently needed.

The object of the present invention was therefore the development of new chemical units which minimize the formation of new aggregates and which can disassemble already existing toxic tau aggregates into native functional tau monomers and thus enable their therapeutic use in different tauopathies.

The chemical unit to be used in therapy or its variants should bind as affinely and specifically as possible to the native, endogenous, monomeric tau protein and thus stabilize it. The balance of misfolded and natively folded tau conformation is thus shifted in favor of the latter. Ideally, already existing tau oligomers and fibrils can be broken down into their monomer building blocks and thus eliminated.

This object was achieved by a peptide according to claim 1, in particular by a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6 or SEQ ID NO: 7 and homologues, fragments and parts thereof. Further preferred embodiments are defined in the dependent claims.

In the following, the term "comprise" should also include "consist of".

The peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6 or SEQ ID NO: 7 were obtained using an optimized mirror image Found phage display selection.

It should be noted that, in addition to the selection of specific monomer binders specified here, the method for mirror image phage display can of course also be used to find specific oligomer binders or even to find ligands for other species occurring in protein misfolding diseases.

In mirror image phage display, e.g. B. a recombinant library of randomized peptide sequences displayed on the gp3 protein of M13 phage and encoded in its genome, selected against the exact mirror image (D-enantiomer) of a naturally occurring L-enantiomeric target molecule (e.g. tau).

The peptide sequence is advantageously presented at the N-terminus of the gp3 protein of M13 phage and is encoded in its genome.

The gp3 molecule, also known as gene product 3, is a protein that is located in the phage coat and is required for contact with the host cell.

The DNA sequence of the p3 gene of a selected phage is linked to the DNA sequence containing the genetic information about the corresponding peptide sequence on the gp3 molecule, allowing it to be sequenced. After sequencing, the genomic sequence can be transcribed into an amino acid sequence and synthesized as a D-enantiomeric peptide that binds to the physiological L-enantiomeric form of the target molecule (e.g., tau). The entirety of the phages which present different peptides as a fusion protein with gp3 on their surface is referred to below as a phage library. The corresponding peptides represent the biomolecules to be selected in the experiment. So-called panning rounds can be carried out, e.g. B. three rounds. The phage library is brought into contact with a fixed target molecule, also known as a bait, and binding phages are isolated from the billions of background other, non-binding phages.

For example, the amount of phage that preferentially bind to oligomeric or fibrillar species of tau is reduced by not offering these species as bait. In this way, phages that show an increased affinity for tau oligomers and fibrils can be removed from the phage pool, so that e.g. B. Tau monomer specific phage enrich. The method can, of course, be adapted in an analogous manner in order to identify ligands and peptides that specifically bind tau oligomers.

In order to reduce accumulation of phages with affinity for plastic, BSA or streptavidin, according to the invention, different substrate surfaces are preferably used in all panning rounds. In this case, a combination of the substrate used (plastic sheet, in this case polystyrene with a streptavidin matrix) and the blocking or quenching agents used is defined as the substrate surface. In the successive selection rounds, the selection pressure is successively increased.

For this purpose, while the concentration of the target molecule (e.g. monomeric tau) remains stable, the number of washing steps after the phage incubation is continuously increased from the 2nd round of selection in order to remove phages that do not have an affinity for tau monomer.

Furthermore, in each selection round of phage display, a different substrate surface is chosen by using different agents to block the surface (e.g., BSA, milk powder, no blocking) after the target molecule has been immobilized on the substrate. For example, you can switch between a milk powder-blocked surface in addition to the biotin-treated surface in round 1, a BSA-blocked surface in round 2 and a milk-powder-treated surface in round 3.

The change between different substrate surfaces increases the specificity for the target molecule or the bait compared to the surface. In addition, there is a reduction in the number of ligands that bind non-specifically to plastic surfaces or the blocking agent.

Parallel to the actual phage display selection, control selections can be carried out, for example, which are related to the implementation are identical to the main selection - with the important difference that no bait is used here. A data analysis of the sequences resulting from the control selections enables the identification of peptides that accumulate during the selection even without decoy and are therefore irrelevant for all subsequent steps.

The method is thus characterized by the following steps: a) Providing an immobilized bait on a substrate surface. b) contacting the baited immobilized molecule with a solution containing a library of molecules. c) Bringing the immobilized bait, which has been occupied with the molecules, into contact with a washing solution. d) Separation and multiplication of the molecules still bound to the bait after bringing the immobilized bait occupied with the molecules into contact with washing solution. e) repeating said steps using a different substrate in each repetition, f) identifying the sequence of the molecules remaining on the bait after the repetition.

A different substrate is z. B. by changing the type of substrate and / or its blocking or non-blocking by means of reagents.

A molecule from the group consisting of proteins, peptides, RNA, DNA, m-RNA and chemical compounds is used as a decoy. In the present case, in particular, monomeric tau protein is used as the bait.

As a surface or substrate to which the bait is immobilized, z. B. used a component from the group consisting of microtiter plates, magnetic particles, agarose or sepharose beads.

The bait according to point a) is therefore a compound to which the biomolecule to be selected is to be bound. It is fixed to a first surface using methods known in the art. Proteins, peptides, RNA or DNA molecules, in particular tau monomer, can be mentioned as decoys by way of example but not by way of limitation. Microtiter plates, magnetic particles, agarose or sepharose beads can be used as possible surfaces, for example. The surface with the immobilized bait can then be quenched, thereby inactivating the functional groups of the substrate. In addition, the free areas remaining on the substrate can be blocked with suitable reagents.

In the second step b), the immobilized bait is brought into contact with a randomized library of molecules, specifically biomolecules. These biomolecules compete to bind to the bait. The randomized library is a mixture of very many, for example 10 ¹² , but also 10 ⁴ or only 100 different molecules in a mixture. Such a library can consist, for example, of peptides, proteins, DNA, RNA or mRNA, which are bound to specific vehicles and which can bind to baits. Examples of suitable vehicles are phages, polysomes or bacterial surfaces. The library can consist of artificial components or components isolated from nature, or a mixture of both. For the purposes of the invention, artificially means, for example, compounds produced from oligonucleotide synthesis.

The immobilized bait covered with biomolecules can be brought into contact with a washing substance in step c). For this purpose, a washing step is carried out in step c), in which a buffer solution is brought into contact with or rinsed with the immobilized baits. This means that the solution with the library of biomolecules is preferably repeatedly exchanged for a possibly similar or identical solution. Thus, those library molecules are removed which dissociate less quickly from the immobilized decoys than other library molecules. The speed of the detachment reaction of the binding library molecules is mainly determined by the different dissociation constants (in particular the k _0ff values) of the individual molecules. From a statistical point of view, those with a small k _0ff value remain bound to the immobilized bait the longest and therefore have a lower statistical probability of being washed away by the wash buffer. The liquid containing the wash buffer is preferably aqueous and may contain a pH buffer. Optional components of the solution for the washing step can be salts, detergents or reducing agents.

After the specificity washing step in step c), the bound biomolecules are separated from the bait and multiplied in step d). The separation in step d) takes place z. B. by eluting phage from the bait. The separation can take place, for example, by changing the pH value, heating or changing, in particular increasing, the salt concentration. In the subsequent multiplication of the library molecules still remaining on the bait, the phage particles obtained, for example, according to steps a) to c) can be introduced into cells and multiplied.

In step e), the concentration of the selected biomolecules in the solution that is fed to the bait after step a) is increased. Preferably 3 to 6 rounds of selection containing steps a) to e) are carried out. However, 1 to 10 or 1 to 20 repetitions can also be carried out. The increase in the competitor concentration in step c), which is preferably carried out here, with an increasing number of cycles also leads to improved selection.

A particularly relevant mirror image phage display provides N-terminally biotinylated D-enantiomeric tau monomer in step a), a recombinant phage library in step b) and a buffer solution in step c) in addition to tau monomer as a bait. An elution as a separation step takes place z. B. via pH reduction as a separation step and phage amplification in bacterial cells as propagation.

In this way, seven D-enantiomeric peptides that bind specifically to tau monomer were selected.

SEQ ID NO 1:

TF2D-1 (free N-terminus, amidated C-terminus): nemylwhwqypqhlrvg

SEQ ID NO 2:

TF2D-5 (free N-terminus, amidated C-terminus): dggyqilfkipgghih

SEQ ID NO 3:

TF2D-la (free N-terminus, amidated C-terminus): nemylwhwqypqhlrvrrrrr

SEQ ID NO 4:

TF2D-lb (free N-terminus; amidated C-terminus): nelylwhwqypqhlrvrrrrr

SEQ ID NO 5:

TF2D-5a (free N-terminus, amidated C-terminus): dggyqilfkipgghihrrrrr

SEQ ID NO 6:

TF2D-5b (free N-terminus, amidated C-terminus): rssyqilfripsshihrrrrr

SEQ ID NO 7:

TF2D-5C (free N-terminus, amidated C-terminus): yqilfripsshihrrrrr

The present invention can also relate to other peptides that can be identified using the method disclosed above.

The peptides according to SEQ ID NO: 1 to 7 can be used as a possible drug against tauopathies such as Alzheimer's dementia due to the specific binding to tau monomers.

The object according to the invention is also achieved by a peptide containing homologues, fragments and parts of the amino acid sequence according to SEQ ID NO: 1 to 7.

Tau peptide or tau protein is preferably understood here to mean the human tau peptide or tau protein.

Homologous sequences" or "homologs" means in the context of the invention that an amino acid sequence has an identity with one of the above amino acid sequences of the monomers of at least 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76 , 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% 80% and 90% are preferred here. Instead of the term "identity", the terms "homologous" or "homology" are used interchangeably in the present description. The identity between two nucleic acid sequences or polypeptide sequences is determined by comparison using the BESTFIT program based on the algorithm of Smith, TF and Waterman, M.S (Adv. Appl. Math. 2:482-489 (1981)) calculated by setting the following parameters for amino acids: gap creation penalty: 8 and gap extension penalty: 2; and the following parameters for nucleic acids: gap creation penalty: 50 and gap extension penalty: 3. The identity between two nucleic acid sequences or polypeptide sequences is preferably defined by the identity of the nucleic acid sequence/polypeptide sequence over the entire sequence length in each case, as determined by comparison using the program GAP based on the algorithm of Needleman, SB and Wunsch, CD (J. Mol. Biol. 48: 443-453) setting the following parameters for amino acids: gap creation penalty: 8 and gap extension penalty: 2; and the following parameters for nucleic acids gap creation penalty: 50 and gap extension penalty: 3.

Two amino acid sequences are identical for the purposes of the present invention if they have the same amino acid sequence.

In one variant, the peptides according to the invention have sequences which differ from the specified sequences by up to two or three amino acids as homologues.

Furthermore, sequences which contain the above-mentioned sequences can also be used as peptides.

The peptides according to the invention are also preferably characterized in that an acid amide group (CONH ₂ group) or a COH group, COCl group, COBr group, CONH-alkyl radical or a CONH- alkyl-amine residue is present or the peptide is present in cyclized form.

In this way, the problem of providing a peptide without a negative charge at the C-terminus is additionally achieved in a particularly advantageous manner. This has the advantageous effect that it can bind to the target molecule with higher affinity than a peptide which has a carboxyl group at the free C-terminus. In the physiological state, peptides with a free, unmodified carboxyl group have a negative charge at this end.

In one embodiment of the invention, the peptides according to the invention are in the physiological state, in particular at pH 6-8, in particular 6.5-7.5, especially at pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5 pH 6.6, pH 6.7, pH 6.8, pH 6.9 , pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9 or pH 8.0 modified such that the C-terminus does not carry a negative charge but instead is neutral or has one or more positive charges.

In one embodiment, the peptide is characterized in that an acid amide group is present at the free C-terminus instead of the carboxyl group. Instead of the carboxyl group (-COOH group), an acid amide group (-CONH ₂ group) is arranged at the C-terminus.

Accordingly, the peptide is particularly advantageously amidated at the free C-terminus and not modified at the free N-terminus.

In this way, the further object is achieved in a particularly advantageous manner, namely that a peptide is present without excess negative charge, which can bind to the target molecule with greater affinity and is easily obtainable.

In a further embodiment of the invention, the following additional groups are present in place of the carboxyl group: COH, COCl, COBr, CONH-alkyl radical, CONH-alkyl-amine radical (net positive charge), etc., without being limited to this, if the technical teaching of the main claim is followed.

In a further preferred embodiment of the invention, the affinity of the binding of the peptides modified according to the invention without a negative charge at the C-terminus is therefore higher by 1%, 2, 3, 4, 5, 6, 7, 8, 9, especially 10%, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,

27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,

69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,

90, 91, 92, 93, 94, 95, 96, 97, 98, 99, especially 100%, 101, 102, 103, 104,

105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,

121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,

137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,

169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,

185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, especially 200%, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,

229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,

245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,

261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,

277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292,

293, 294, 295, 296, 297, 298, 299, especially 300%, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,

322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337,

338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,

354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,

370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385,

386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, especially 400%, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428,

429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,

445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460,

461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476,

477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,

493, 494, 495, 496, 497, 498, 499, advantageously even 500%, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516 , 517, 518, 519, 520,

521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536,

537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552,

553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568,

569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584,

585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, particularly advantageous 600%, 601, 602, 603, 604, 605, 606, 607, 608 , 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626,

627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642,

643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658,

659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674,

675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690,

691, 692, 693, 694, 695, 696, 697, 698, 699, particularly advantageously 700%, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714 , 715, 716, 717,

718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749,

750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765,

766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781,

782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797,

798, 799, also particularly advantageous 800%, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822,

823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838,

839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854,

855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870,

871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886,

887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, also particularly advantageous 900%, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926,

927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942,

943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958,

959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974,

975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990,

991, 992, 993, 994, 995, 996, 997, 998, 999, or even increased by 1000%, or even by 10000%, or even by up to 100000% or 1000000%, any intermediate value can be accepted.

This is indicated by a correspondingly reduced Kü value. The Ku value, as a measure of the binding affinity of a modified peptide to tau monomer, is increased by 1%, 2, 3, 4, 5, 6, 7, 8, 9, especially 10%, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,

35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,

56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,

77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,

98, 99, especially 99.1, 99.2, 99.3, 99.4, 99.5%, 99.6, 99.7, 99.8, 99.9 up to 99.99 or even 99.999% reduced, whereby any intermediate value can be assumed.

The peptide according to the invention is further preferably characterized in that it contains 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the sequences with the SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO : 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and/or SEQ ID NO: 7. Variants are also conceivable, with the peptide 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more peptides with the SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 , SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and/or SEQ ID NO: 7.

Dimers of the sequences with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6 and/or SEQ ID NO: 7 are particularly preferred , where the two monomers of the dimer are peptides with the same SEQ ID or with a different SEQ ID.

The peptides according to the invention are further preferably characterized in that they consist essentially of D-amino acids.

For the purposes of the present invention, the term "essentially composed of D-enantiomeric amino acids" means that the peptides to be used according to the invention contain at least 50%, 55%, 60%, 65%, 70%, preferably 75%, 80%, particularly preferably 85%, 90%, 95%, in particular 96%, 97%, 98%, 99%, 100% are made up of D-enantiomeric amino acids.

The peptide according to the invention is further preferably characterized in that it consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6 or SEQ ID NO: 7.

The peptide according to the invention is also preferably characterized in that the peptide is linked to another substance.

Within the meaning of the invention, the linkage is a chemical bond such as that described in Römpp Chemie Lexikon, 9th edition, volume 1, page 650 et seq.

Georg Thieme Verlag Stuttgart is defined, preferably around a main valence bond, in particular a covalent bond.

In one variant, the substances are drugs or active ingredients, defined in accordance with the Drugs Act §2 or. §4 (19),

As of September 2012. In an alternative, active substances are therapeutically active substances that are used as medicinally active substances. Anti-inflammatory drugs are preferred. In a further variant, the substances are compounds which enhance the effect of the peptides.

Another alternative involves compounds that improve the solubility of the peptides and/or the passage of the blood-brain barrier.

In an alternative, the peptides according to the invention have any combination of at least two or more features of the variants, embodiments and/or alternatives described above.

The peptide according to the invention is also preferably characterized in that a plurality of peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID

NO: 4, SEQ ID NO: 5 SEQ ID NO: 6 or SEQ ID NO: 7 are covalently or non-covalently linked to one another.

A covalent connection or linkage of the peptide units is present within the meaning of the invention if the peptides are linearly linked head to head, tail to tail or head to tail, with or without linkers or linker groups inserted in between.

NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 without a linker, ie linked directly to one another, or are linked to one another with a linker group.

A non-covalent linkage within the meaning of the invention is present if the peptides are linked to one another, for example via biotin and streptavidin, in particular streptavidin tetramer.

The peptide according to the invention is also preferably characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6 or SEQ ID NO: 7 are linked to one another in a linear or branched manner.

In a variant of the present invention, the peptides can be linked to one another in a linear manner, in particular as described above. In another Variant, the peptides are branched together to form the peptide according to the invention.

According to the invention, a branched peptide can be a dendrimer in which the monomers are linked to one another covalently or non-covalently.

Alternatively, the peptides can also be linked to a platform molecule (such as PEG or sugar) to form a branched peptide.

Alternatively, combinations of these options are also possible

In one embodiment of the invention, the affinity of the binding of the peptides is defined via the dissociation constant (Ku value).

The dissociation constant (Kü value) of a peptide according to the invention is advantageously reduced in an advantageous embodiment of the invention. Associated with this are improved properties of the peptides according to the invention, such as higher binding affinity and higher effectiveness of the degradation and/or prevention of the formation of toxic tau oligomers or aggregates.

In a variant of the invention, such peptides are used which are attached to a tau monomer with a dissociation constant (Kü value) of at most 500 pM, preferably 250, 100, 50 pM, particularly preferably 25, 10, 1 pM, particularly preferably with a Dissociation constant (Kü value) of at most 500 nM, 250, 100, 50, particularly preferably 25, 10, 1 nM, 500 pM, 100, 50, 25, 20, 15, 10, 9,

8, 7, 6, 5, 4, 3, 2, 1 pM to sub-pM binds, any intermediate value can be assumed.

The peptides according to the invention are also preferably characterized in that they bind to the tau protein, preferably the natively folded tau protein, with a K _D of less than 1 pM.

The peptides can be produced, for example, via chemical synthesis or peptide synthesis. Another variant is a peptide according to the invention for inhibiting or preventing the formation of tau peptide oligomers and/or tau peptide aggregates.

Another variant is a peptide according to the invention for the detoxification of tau peptide oligomers and/or tau peptide aggregates.

The peptides according to the invention detoxify the tau peptide oligomers and/or tau peptide aggregates or polymers formed therefrom, as well as fibrils, preferably by not binding to them but by binding to tau monomer and by shifting the equilibrium leading to the reduction of the tau oligomers and these thus converted into non-toxic compounds. Accordingly, the subject matter of the present invention is also a method for detoxifying the tau oligomers, aggregates or fibrils formed therefrom.

The inhibition or prevention of the formation of tau peptide oligomers and/or tau peptide aggregates, or the detoxification of the tau peptide oligomers and/or tau peptide aggregates can take place in vitro or in vivo.

The present invention also relates to a peptide according to any one of the preceding claims for use in the treatment of tauopathies.

The present invention also relates to a peptide according to any one of the preceding claims, in particular for use in the treatment of Alzheimer's dementia (Alzheimer's).

The invention is explained in more detail below in non-limiting examples. examples

The peptides TF2D-1, TF2D-5 and TF2D-5a were examined in more detail.

The results are summarized in FIGS. 1, 2a, 2b, 3, 4, 5 and 6.

Figure 1: TF2D-1 and TF2D-5 inhibit tau aggregation analyzed by ThT analysis

Shown are the effects of TF2D-1 and TF2D-5 on tau aggregation. The ThT assay was carried out by incubating 15 pM tau with 8 pM fleparin, 20 pM TFIT and 15 pM one of the two peptides TF2D-1 or TF2D-5 at 37° C. in PBS pH 7.4. The sample without the addition of the peptide is shown in red. The samples in which TF2D-1 or TF2D-5 was present are shown in green (TF2D-1) and blue (TF2D-5).

Figure 2a: TF2D-5 binds to tau protein with high affinity

SPR measurements of TF2D-5 with the analyte Tau. Seven concentrations of tau were prepared for the measurement, with the highest concentration being 100 nM and the lowest being 1.56 nM. The running buffer contained TBS with 0.1% Tween. The Koff was determined to be about 6x10-5 s-1.

Figure 2b: TF2D-5 and TF2D-5a bind to the tau protein with high affinity

SPR measurement of TF2D-5 and TF2D-5a with full-length tau 441 A) The binding curves and the calculated fit for the affinity of TF2D-5 for monomeric full-length tau 441 revealed a KD of 1.3 nM. The tau concentration during the measurement varied from 3.125 nM to 25 nM. B) The binding affinity of TF2D-5a to full-length tau was determined to be 6.1 nM. Tau was used at concentrations from 31.25 nM to 500 nM. The SPR measurements of A and B were recorded on a T200 Biacore instrument, the respective peptide was immobilized on a Cytiva CM5 chip, while monomeric tau served as the analyte. The tests were performed with TBS pH 7.4, 0.05% Tween 20 and 10 pM DTT as running buffer. The fit calculation was performed with the internal evaluation software of the T200 Biacore. A l:l model was used for both fits. Figure 3: TF2D-5 inhibits tau aggregation analyzed by ThT and AFM analysis

Shown are the effects of TF2D-5 on tau aggregation. The ThT assay was performed by incubating 15mM tau with 8mM fleparin, 20mM ThT, 10% DMSO and various concentrations of TF2D-5 at 37°C pH 7.0. The images next to the graphic show the respective AFM measurements of the individual samples, which were carried out after 5 days. Shown in A is the sample that did not contain TF2D-5. Figure 6 shows the sample of A with a 1:10 dilution. The image marked C corresponds to the blue ThT curve without dilution with TF2D-5 added after 24 hours. All AFM measurements were performed by drying 1mI on a mica, followed by two washing steps with 100mI water and measurement on JPK Nanowizard3 with intermittent contact air and an OMCL AC160TS as cantilever.

Figure 4: TF2D-5 and TF2D-5a affinity to tau is sequence specific

MST measurement of TF2D-5, TF2D-5a and CP1 with full length rope. A) Boltzmann fit of TF2D-5 MST measurements with full-length tau. TF2D-5 was tagged CF633. The concentration of TF2D-5 was 500 nM, while the concentration of full-length tau ranged from 2.5 mM to 0.2 pM. The measurement was carried out in 1x PBS pH 7.4 with 150 mM NaCl. The calculated KD is 308 pM +/- 148 pM. B) Boltzmann fit of TF2D-5a MST measurements with full-length tau. TF2D-5a was labeled CF633. The concentration of TF2D-5a was 62.5 nM while the concentration of full-length tau varied between 300 nM and 0.5 nM. The measurement was carried out in 0.1 M sodium phosphate buffer pH 7.4 with 75 mM sodium sulfate and 0.05% Tween 20. The calculated KD is 11.7 nM +/- 1.3 nM. C) Average of CPl-MST measurements with full-length tau. CP1 consists of the same amino acids as TF2D-5, but with a different amino acid distribution. CP1 was labeled CF633. The concentration of CP1 was 62.5 nM while the concentration of full-length tau varied between 2.5 mM and 0.2 pM. The measurement was carried out in 0.1 M sodium phosphate buffer pH 7.4 with 75 mM sodium sulfate and 0.05% Tween 20. No Boltzmann fit and no KD could be calculated for the tested concentration range. D) Overview of calculated KDS of the individual peptides to full-length tau. All experiments were repeated four times and the average of all measurements was calculated, which in turn was used to calculate the Fit's (Boltzmann).

Figure 5: TF2D-5a inhibits aggregation of tau in cells.

A) TF2D-5a and the D-peptide CP-1, as a negative control, were tested for their ability to inhibit tau aggregation in tauK18(LM)-YFP cells. The scale bar corresponds to 50 pm and applies to b-d. Significance was calculated using a one-way ANOVA followed by Dunnett's test for multiple comparisons. Three asterisks represent a p-value less than 0.001 and four asterisks represent a p-value less than 0.0001. B) In contrast to unseeded cells, which showed no tau aggregates on the third day after seeding, seeding with sonicated tau fibrils resulted in aggregation in about 40% of the cells after three days of incubation. C) Three-day treatment with the D-peptide CP-1 as a negative control did not inhibit tau aggregation. D) In contrast, treatment with increasing concentrations of TF2D-5a resulted in a concentration-dependent reduction in the number of cells with tau aggregates, which was significant at 12.5 pM and was further reduced with 25 pM and 50 pM TF2D-5a.

Figure 6: TF2D-5 and TF2D-5a are very stable in different body fluids

Stability study of TF2D-5 and TF2D-5a in various simulated human body fluids. A) Stability of the peptides in simulated gastric fluid (SGF). The simulated gastric fluid consisted of 2 mg/ml sodium chloride, 3.2 mg/ml pepsin and 80 mM FICI; pH 1. B) Stability of TF2D-5 and TF2D-5a in simulated intestinal fluid (SIF). Simulated intestinal fluid consisted of 6.8 mg/ml potassium dihydrogen phosphate, 10 mg/ml pancreas powder and 15.4 mM NaOFI; pH 6.8. C) Stability of TF2D 5 and TF2D-5a in human blood plasma. Flumanes K3EDTA blood plasma was obtained from BioTrend. D) Stability of TF2D-5 and TF2D-5a in human liver microsomes. Human liver microsomes consisted of 6 mg/ml protein (which contains cytochrome P450 (CYP) enzymes), 75 mM sucrose, 1.3 mM NADPH, 3.3 mM D-glucose-6-phosphate, 0.2 u/ml D - Glucose-6-phosphate dehydrogenase, 3.3 mM magnesium chloride and 100 mM potassium phosphate pH 7.4.

Claims

Expectations

1. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, as well as homologues, fragments and parts thereof.

2. Peptide according to claim 1, characterized in that the peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6 or SEQ ID NO: 7 or homologs with at least 80% identity thereof.

3. Peptide according to any one of the preceding claims, characterized in that at the free C-terminus instead of the carboxyl group an acid amide group (CONH ₂ group) or a COH group, COCl group, COBr group, CONH-alkyl radical or a CONH-alkyl-amine residue is present or the peptide is present in cyclized form.

4. Peptide according to any one of the preceding claims, characterized in that there are 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copies of the sequences with the SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

5. Peptide according to any one of the preceding claims, characterized in that the peptide consists essentially of D-amino acids.

6. Peptide according to any one of the preceding claims, characterized in that the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6 or SEQ ID NO: 7.

7. Peptide according to any one of the preceding claims, characterized in that the peptide is linked to another substance.

8. Peptide according to any one of the preceding claims, characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 are covalently or non-covalently linked to one another.

9. Peptide according to any one of the preceding claims, characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6 or SEQ ID NO: 7 without a linker, ie linked directly to one another, or are linked to one another with a linker group.

10. Peptide according to any one of the preceding claims, characterized in that several peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6 or SEQ ID NO: 7 are linked to one another in a linear or branched manner.

11. Peptide according to any one of the preceding claims, characterized in that it is a dendrimer, wherein peptides of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO : 5 SEQ ID NO: 6 or SEQ ID NO: 7 are linked to a platform molecule.

12. Peptide according to any one of the preceding claims, characterized in that it binds to the tau peptide with a K _D of less than 1MM.

13. Peptide according to any one of the preceding claims for preventing the formation of tau peptide oligomers and/or tau peptide aggregates.

14. Peptide according to any one of the preceding claims for the detoxification of tau peptide oligomers and/or tau peptide aggregates.

15. A peptide according to any one of the preceding claims for use in the treatment of tauopathies.